Several studies have shown that recombinant FVIII products often have discrepant potencies when different assay methods are applied. In the case of one recombinant product, use of the chromogenic assay, mandated in Europe, would mean that 30% less factor VIII would be delivered to patients on a molar basis than if one applied the standard one-stage clotting assay used in the United States. In contrast, a B-domain deleted recombinant FVIII gave about 1.25 fold higher potency in the one-stage clotting assay than in the chromogenic assay. It is important for us to discern the mechanism responsible for such discrepancies, to achieve standardization of potency measurements for recombinant FVIII. Initial approaches: The activity of FVIII from six different recombinant and plasma-derived FVIII preparations were examined with either one-stage clotting or chromogenic assays. We found that one recombinant FVIII product had discrepancy in its activity when assayed by the two different assay methods. The chromogenic assay gave significantly higher activity than the one-stage clotting assay (Chromogenic/one-stage=1.445, n=7, 0.001>p>0.0001). By immunoprecipitation and Western-blotting analysis, we found that this FVIII had more polypeptide species that contained the B-domain of FVIII compared to plasma-derived FVIII. In addition, we found a >200 kDa band not observed in plasma-derived FVIII. Antibody mapping suggests that the >200 kDa polypeptide is an N-terminal truncated single polypeptide FVIII. Future studies: The bioactivity of the B-domain in FVIII is not fully understood. Proteolytic digestion of the FVIII light chain is important for the specific activity of FVIIIa and thrombin digestion of the light chain is much faster in the B-domain deleted form of FVIII. We will evaluate the effect of the B-domain on the proteolytic digestion of the FVIII light chain by Factor Xa. This study is important and relevant because Factor Xa is the primary protease to activate FVIII in chromogenic assay, while thrombin is the primary protease to activate FVIII in one-stage clotting assay. We will also evaluate the effect of the N-terminal truncated >200 kDa FVIII or other proteins, such as von Willebrand Factor, on the FVIII activity assays.